A critical step in HIV-1 replication is the disassembly (uncoating) of the viral core. Remarkably, the timing and intracellular location of HIV-1 uncoating remain unknown. Studies of HIV-1 uncoating have been hampered by an inability to accurately quantify capsid protein (CA) loss from the viral complexes and by an inability to identify rare infectious viral complexes (~1/50) in infected cells.
We developed methods to label CA with GFP (GFP-CA) in infectious viral complexes and to identify transcriptionally-active proviruses in live-cell imaging assays. We analyzed the dynamics of viral complex association with nuclear envelope and nuclear uncoating, and identified rare viral complexes that integrate to form transcriptionally active proviruses.
Using live-cell imaging, we observed >110 GFP-CA-labeled infectious viral complexes that integrated and expressed HIV-1 RNA and the gfp reporter gene. The infectious viral complexes maintained steady GFP-CA fluorescence signals for several hours after nuclear import followed by abrupt (<20 min) GFP-CA loss ~10.5 hours after infection, signifying nuclear uncoating. HIV-1 transcription sites appeared near the sites of nuclear uncoating, indicating that uncoating occurs at or very close to the site of integration. Similar GFP-CA fluorescence intensities of nuclear viral complexes and viral cores in vitro suggest that viral cores in the nucleus retain >90% of the CA and that nuclear uncoating is the major uncoating event. The nuclear GFP-CA-labeled viral complexes rapidly disassembled after treatment of the infected cells with capsid inhibitor PF74 indicating that the nuclear viral complexes retained CA hexamers. Time-of-addition assays with PF74, nevirapine, and raltegravir indicate that nuclear uncoating occurs ~3 hrs after the completion of reverse transcription and ~1 hr before integration. We probed the potential mechanism by which viral cores enter the nucleus and found that cleavage and polyadenylation specificity factor 6 (CPSF6), a host nuclear protein that binds to CA, influences the intracellular location of uncoating and facilitates the nuclear import of intact or nearly intact viral cores.
Intact or nearly intact viral cores of infectious viral complexes that retain >90% of their CA enter the nucleus and uncoat near their genomic integration sites just before integration.